Neoepitope vaccine and immune stimulant combinations and methods

ABSTRACT

Cancer is treated via a coordinated treatment regimen that use various compounds and compositions that employ prime-boost vaccination in combination with immune modulatory treatment and biasing of an immune response towards a Th1 profile.

This application is a continuation of U.S. Pat. Application No.16/390,946, filed Apr. 22, 2019, which in turn claims priority to U.S.Provisional Pat. Application No. 62/661,298, filed Apr. 23, 2018. Bothof these applications are incorporated by reference in its entiretyherein.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of cancertherapy, especially as it relates to protocols and compositions forcancer immunotherapy that utilize vaccine and immune modulators in acoordinated manner.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

The immune system has been described as playing a dual role in cancer asit can protect against cancer development by detecting and eliminatingtumor cells, but also in that it can promote cancer progression byselection of tumor cells that escape immune destruction. Thisparadoxical role of the immune system in cancer is also known as cancerimmunoediting (see e.g., Science. 2011;331:1565-70). Cancer cellsfrequently employ various mechanisms to evade recognition anddestruction by immune cells. For example, cancer cells can modulate thetumor microenvironment (TME) through recruitment of regulatory T cells(Tregs), myeloid-derived suppressor cells (MDSCs), and immunosuppressivemacrophages (M2 macrophages). Cancer cells can also evade the immunesystem by down-regulating expression of certain MHC (majorhistocompatibility complex) molecules, which are deemed typicallyessential for T cells to recognize tumor-associated antigens (TAAs).Still further, cancer cells can secrete immune suppressive cytokines(e.g., TGF-β, IL-8) to establish a more immune suppressive environment.

To overcome at least some of the downregulating functions in the immuneevasion of a tumor, various pharmaceutical agents have been tested withvarying degree of success. For example, immune stimulants can beadministered to increase an immune response to specific tumor antigens.Among other immune stimulants, certain cytokines and derivatives areoften used and include IL-2, IL-12, or IL-15, and chimeric forms thereofsuch as ALT-803 (see e.g., Altor BioScience Corporation, 2810 NorthCommerce Parkway, Miramar, FL 33025; ALT-803 FACT SHEET) or NHS-IL-12(see e.g., Oncotarget 2014, Vol.5, No.7, 1869-1884). While increasingsome of the parameters in an immune response, administration of suchcompounds per se has not been proven to be therapeutically effective ineradication of a tumor. In yet another known approach, variouscheckpoint inhibitors have been used in the treatment of cancer, and insome cases yielded remarkable results. However, as with thecytokine-type immune stimulants, consistent responses have been elusive,especially across different types of cancer. In other known approaches,tumor vaccines have been employed to generate an immune response againsta tumor specific or tumor associated antigen. Once more, notable immuneresponses were observed in some cases. However, immune stimulation withvaccine compositions is still often associated with suppression of animmune response in a tumor. To improve immune response and reduce immunesuppression, timed immunotherapy has been described to address immuneediting as taught in WO 2018005973. Such coordinated therapyadvantageously addresses immune escape from various angles. However,such therapy is often lengthy and requires multiple treatment agents anda complex treatment regimen.

Consequently, there is still a need to provide treatment compositionsand methods that improve cancer immune therapy, and especially a therapythat is conceptually simple and effective using a minimum of therapeuticagents.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various compositions andmethods of cancer immune therapy, in which various pharmaceuticalcompositions are administered to the patient to so elicit atherapeutically effective response. Most preferably, such methodsinclude prime/boost administration of a cancer vaccine with concurrentand overlapping immune stimulation and checkpoint inhibition regimes. Itis further preferred that the so generated immune response is furthermodified/enhanced by administration of a pharmaceutical that triggers aTh1 bias of the immune response.

In one aspect of the inventive subject matter, the inventors contemplatea method of stimulating an immune response in an individual against a(typically solid) tumor. The method includes a step of subjecting theindividual to a timed treatment combination that includes a prime-boostvaccination regime, an immune stimulation regimen, a checkpointinhibition regimen, and a Th1 stimulation. Most preferably, the immunestimulation regimen commences after the prime vaccination, thecheckpoint inhibition regimen commences after the immune stimulationregimen, and the Th1 stimulation commences after the boost vaccinationor subsequent administration of the vaccine composition.

While not limiting to the inventive subject matter, it is contemplatedthat the prime-boost vaccination regime comprises administration of avaccine composition that elicits an immune response towards at least oneof a tumor associated antigen and a tumor specific antigen, and it isgenerally preferred that the vaccine composition comprises a bacterial,a yeast, or a viral vaccine composition. In further typical embodiments,the prime-boost vaccination regime comprises administration of thevaccine composition in weekly intervals.

It is further contemplated that the immune stimulation regimen comprisesadministration of at least one of IL-2, IL-12, IL-15, IL-21 or aderivative thereof (which may include a targeting antibody, or anantibody fragment), and an especially preferred derivative is ALT-803.For example, the immune stimulation regimen may comprise administrationof at least one of IL-2, IL-12, IL-15, IL-21 or a derivative thereof in3-5 day intervals. With respect to checkpoint inhibition it iscontemplated that the checkpoint inhibition regimen comprisesadministration of an anti PD1 antibody, an anti-PD-L1 antibody, and/oran anti-CTLA4 antibody, which is preferably performed in 1-3 dayintervals.

Contemplated Th1 stimulations typically comprise administration of acytokine or small molecule drug that biases an immune response towards aTh1 response. Therefore, especially preferred cytokines include IL-12 ora derivative thereof (e.g., comprising an antibody portion that binds toa tumor cell or a necrotic component of a tumor cell).

In further contemplated aspects, the immune stimulation regimen and thecheckpoint inhibition will regimen overlap, and/or the immunestimulation regimen, the checkpoint inhibition regimen, and the Th1stimulation are at least initiated during the prime-boost vaccinationregime.

In another aspect of the inventive subject matter, the inventorscontemplate a method of treating a tumor in an individual that includesthe following steps: (a) subjecting the individual to a vaccinationregimen with a vaccine composition to elicit an immune response towardsat least one of a tumor associated antigen and a tumor specific antigen,wherein the vaccination regimen comprises at least a first and a secondadministration of a vaccine composition; (b) subjecting the individualto an immune stimulation regimen after the first administration of thevaccine composition, wherein the immune stimulation regimen comprises atleast a first and a second administration of an immune stimulatorycomposition; (c) subjecting the individual to a checkpoint inhibitionregimen after the first administration of the immune stimulatorycomposition, wherein the checkpoint inhibition regimen comprises atleast a first and a second administration of a checkpoint inhibitor; and(d) subjecting the individual to Th1 stimulation after the second orsubsequent administration of the vaccine composition, wherein the Th1stimulation comprises administration of a compound that biases an immuneresponse towards a Th1 response.

Most typically, the first and the second administration of the vaccinecomposition, and optionally the second administration and a subsequentadministration of the vaccine composition, are between 5 and 10 daysapart, and/or the first administration of the immune stimulatorycomposition is between the first and second administration of thevaccine composition. It is further contemplated that the firstadministration of the checkpoint inhibitor is between the first andsecond administration of the immune stimulatory composition, orconcurrent with or after administration of the second administration ofthe vaccine composition, and/or that the immune stimulation regimen andthe checkpoint inhibition regimen are performed between the first and alast administration of the vaccine composition. Therefore, the immunestimulation regimen and the checkpoint inhibition regimen may overlap.In further contemplated embodiments, the first and the secondadministration of the immune stimulatory composition are between 3-5days apart, and/or the first and the second administration of thecheckpoint inhibitor are between 1-3 days apart. As noted above, it isalso contemplated that the compound that biases the immune responsetowards the Th1 response further comprises an antibody or antibodyfragment that binds to a tumor cell or a component of a necrotic cell.

Consequently, it should be appreciated that the inventors alsocontemplate a coordinated use of a vaccine composition, an immunestimulatory composition, a checkpoint inhibitor, and a compound thatbiases an immune response towards a Th1 response. In such use, it isgenerally preferred that the immune stimulatory composition isadministered after administration of a first dose of the vaccinecomposition, that the checkpoint inhibitor is administered after a firstdose of the immune stimulatory composition, and that the compound thatbiases the immune response towards the Th1 response is administeredafter a second or subsequent administration of the vaccine composition.

Preferably, the vaccine composition elicits an immune response towardsat least one of a tumor associated antigen and a tumor specific antigen,and/or comprises a bacterial vaccine, a yeast vaccine, or a viralvaccine. Moreover, it is generally preferred that the immune stimulatorycomposition comprises a cytokine or a cytokine derivative (e.g., atleast one of IL-2, IL-12, IL-15, IL-21 or a derivative thereof where thederivative thereof comprises a targeting antibody, or an antibodyfragment, and/or ALT-803). Optionally, the checkpoint inhibitorcomprises an antibody binding to PD1, PD-L1, or CTLA4. Furtheroptionally, it is generally preferred that the compound that biases theimmune response towards the Th1 response comprises IL-12, optionallycoupled to an antibody or fragment thereof that binds to a tumor cell ora component of a necrotic tumor cell (e.g., NHS IL-12). In furthercontemplated uses, the vaccine composition is administered using aweekly schedule, the immune stimulatory composition is administeredusing a 3-5 day schedule, and the checkpoint inhibitor is administeredusing a 1-3 day schedule.

Various objects, features, aspects, and advantages of the inventivesubject matter will become more apparent from the following detaileddescription, along with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic illustration of a personalized vaccine preparationused in the inventive subject matter.

FIG. 2 is a graph depicting exemplary results for patient neoepitopefiltering using expression and calculated MHC binding.

FIG. 3 is an exemplary schematic of various control experiments comparedwith a vaccination scheme according to the inventive subject matter.

FIG. 4 depicts photomicrographs of CD8 T cell infiltration of tumors inanimals of the schemes or FIG. 3 .

FIG. 5 is an exemplary graph of tumor volumes in animals treated using ascheme according to the inventive subject matter.

FIGS. 6A-H show the procedures for identification of immunogenicneoepitopes. FIG. 6A shows workflow for neoepitope discovery insubcutaneously implanted MC38 tumors. FIG. 6B shows in-vitro bindingscore of MC38 neoepitopes. FIG. 6C shows IFNγELISPOT analysis of naivemice vaccinated twice with pools of 9-mer neoepitopes. Splenocytes wereharvested 1 week following the second vaccination. Shaded bars representpeptides that induced robust immune responses in repeated experiments.FIG. 6D shows treatment schedule. FIGS. 6E-F shows day 18 IFNγELISPOTanalysis of mice vaccinated with (6E) four 9-mer or (6F) two 25-merneoepitopes, alone or in combination with N-803 and/or anti-PD-L1. FIG.6G shows survival curves of tumor-bearing mice treated with N-803,anti-PD-L1 and four 9-mer neoepitope peptides (black line), N-803,anti-PD-L1 and PBS (dashed line), or no treatment (gray line). FIG. 6Hshows survival curves of mice treated with 9-mer neoepitope vaccine,N-803 and anti-PD-L1 stratified by antigen- specific IFNγ-secretingcells per 106 cells in the peripheral blood on day 13, *P<0.05.

FIGS. 7A-H show combination therapy using a 9-mer neoepitope vaccine,N-803, anti-PD-L1 and NHS- IL12. FIG. 7A shows treatment schedule. FIG.7B shows IFNγ ELISPOT analysis on days 11, 18 and 25 of tumor growthagainst peptides contained within the vaccine (top) or MC38 neoepitopesnot contained within the vaccine or P15e (bottom). Each columnrepresents one mouse. FIG. 7C shows tumor growth curves. FIG. 7D showstumor growth in mice treated with 9-mer neoepitope vaccine, N-803,anti-PD-L1 and NHS-IL12 stratified by antigen-specific IFNγ-secretingcells per 106 cells in the peripheral blood on day 13. FIG. 7E showssurvival curves after re-challenge of naive animals or those with apreviously regressed MC38 tumor following indicated treatment.Re-challenged animals were implanted with MC38 tumors on day 0 ofsurvival curve and received no subsequent therapies. Arrow indicatesdepletion of CD8+ cells. FIGS. 7F-G show analysis of splenocytes frommice treated with 9- mer neoepitope vaccine, N-803, anti-PD-L1 andNHS-IL12, harvested on day 25 and stimulated overnight with eithervaccine components or cascade antigens. FIG. 7F shows ELISPOT analysisof the ratio of antigen- dependent TNFα:IFNγ secreting splenocytes. FIG.7G shows flow cytometric analysis of percent of CD8+ cells that weresingle IFNγ(orange), single TNFα (pink), or double (purple) producers.FIG. 7H shows Tumor growth in mice treated with 9-mer neoepitope vaccineand NHS-IL12 according to the schedule in FIG. 7A.

FIGS. 8A-B show tumor progression in mice treated with combinationtherapy using single-peptide vaccines or immune cell depletions. FIG. 8Ashows tumor growth in mice treated with N-803, anti-PD-L1, NHS-IL12 anda neoepitope vaccine consisting of a single 9-mer neoepitope or a poolof four 9- mer neoepitopes. Mice were treated according to the schedulein FIG. 7A. FIG. 8B shows timeline (left) and tumor growth (right) ofdepletion studies. Tumor-bearing mice were depleted of NK, CD4 or CD8cells starting 3 days prior to the first vaccine (early depletion) orNHS-IL12 administration (late depletion).

FIGS. 9A-D show tumor progression and tumor-infiltrating immune cells inmice treated with single, triple, or quadruple combinations of N-803,anti-PD-L1, NHS-IL12 and 9-mer neoepitope vaccine. Mice were treated aspreviously described, and tumors were harvested on day 22 post- tumorimplantation. Tumors were analyzed via (9A+9B) immunofluorescentanalysis or (9C+9D) flow cytometry. FIG. 9A shows representativeimmunofluorescent images of CD8+ (red) cells in zinc formalin-fixedparaffin-embedded tumors sections. Blue corresponds to DAPI staining.Scale bar= 50 µm. FIG. 9B shows Percent CD8+ cells (of total DAPI+cells) in immunofluorescent sections. FIG. 9C shows intratumoral M1macrophages (top, CD1 1b+F4/80+CD38+) and M2 macrophages (CD11b+F4/80+CD206+). FIG. 9D shows CD8+ TIL maturation (CD44/CD62L/CD127).*P<0.05, **P<0.01, ***P<0.001, **** P<0.0001.

FIGS. 10A-D show Gene expression and clonality of tumor-infiltratingimmune cells after treatment with a 9-mer neoepitope vaccine, N-803,anti-PD-L1 and NHS-IL12. FIG. 10A shows gene expression analysis oftumor-infiltrating leukocytes. FIG. 10B shows clonality of TCRβchainsdetected in tumor infiltrates **P<0.001. FIG. 10C shows number ofTCRβclones that comprise the top 25% of detected sequences. FIG. 10Dshows frequency of the top 100 TCRβ sequences detected in each sample.Each column represents one mouse, and each row represents a uniqueclone.

FIGS. 11A-K show combination therapy utilizing adenoviral vectors. FIG.11A shows Visual representation of admixed and multi-epitope adenoviralvaccines targeting neoepitopes. FIG. 11B shows treatment schedule. FIG.11C shows IFNγ ELISPOT analysis on day 25 of tumor growth againstneoepitopes contained within the vaccine. Each row represents one mouse.FIG. 11D shows immune responses generated in animals vaccinated withmulti-epitope adenovirus relative to paired neoepitopes from animalstreated with admixed adenovirus. Each row represents one mouse. FIG. 11Eshows tumor growth curves. FIG. 11F shows representativeimmunofluorescent images of CD8+ (red) cells in zinc formalin-fixed,paraffin-embedded tumors sections. Blue corresponds to DAPI staining.Scale bar = 50 µm. FIG. 11G shows ratio of CD8:CD4 TIL inimmunofluorescent sections. FIG. 11H shows % FoxP3+ cells withinimmunofluorescent sections. FIG. 11I shows tumor growth in mice treatedas indicated in FIG. 11B utilizing an adenovirus targeting TWIST1. FIG.11J shows IFNγ ELISPOT analysis on day 25 of tumor growth againstneoepitopes identified in the MC38 cell line. Each column represents onemouse. FIG. 11K shows relative immunity of the immune responsesgenerated in animals against neoepitopes in the adeno multi-epitopevaccinated animals as compared to animals vaccinated with adeno-TWIST1vaccine. Each column represents one mouse.

DETAILED DESCRIPTION

The inventors have discovered that cancer immune therapy can beadministered using a coordinated orchestration of a combination ofcertain immunotherapeutic agents to so elicit an effective anti-tumorresponse in a mammal. Preferably, such orchestrated treatments are basedon a prime/boost vaccination regimen that includes an immune stimulationphase using one or more immune stimulatory cytokines and checkpointinhibition phase using one or more checkpoint inhibitors, before the soelicited immune response is further maintained and/or modified towards aTh1 response.

Thus, it is also contemplated that immune therapy using a prime/boostregiment with cancer vaccine can be significantly improved bystimulating the immune system after the prime vaccination with immunestimulatory cytokines, and by further modulating suppressive signalswith checkpoint inhibitors, preferably at or during the boost phase ofthe vaccination. Stimulation and checkpoint inhibition are continuedduring the boost phase, and the so elicited immune response is thensupported and/or further modulated towards a Th1 response byadministration of a second immunostimulatory cytokine, preferably in atumor targeted manner.

Notably, such protocol provided significant therapeutic effect as isfurther demonstrated in more detail below. As the immunomodulation incontemplated treatments are independent of the particular tumor type,and as the vaccine is specific to a patient’s specific tumor, it shouldbe recognized that contemplated compositions and methods will besuitable for a variety of tumors, including solid tumors, soft tissuetumors, and lymphatic tumors.

As used herein, the term “tumor” refers to, and is interchangeably usedwith one or more cancer cells, cancer tissues, malignant tumor cells, ormalignant tumor tissue, that can be placed or found in one or moreanatomical locations in a human body. As used herein, the term “bind”refers to, and can be interchangeably used with a term “recognize”and/or “detect”, an interaction between two molecules with a highaffinity with a K_(D) of equal or less than 10⁻⁶ M, or equal or lessthan 10⁻⁷ M. As used herein, the term “provide” or “providing” refers toand includes any acts of manufacturing, generating, placing, enabling touse, or making ready to use.

For example, with respect to the vaccination regimen it should beappreciated that the type and content of the vaccine composition toelicit an immune response towards at least one of a tumor associatedantigen and a tumor specific antigen may vary considerably.Consequently, all manners of providing an immunogen to an individual aredeemed suitable and include DNA vaccines, peptide vaccines, andespecially recombinant vaccines. Likewise, the content of the vaccinesmay vary considerably and may include a tumor associated antigen orfragment thereof (e.g., MUC1, brachyury, CEA, etc.), a tumor specificantigen or fragment thereof (e.g., PSA, PSMA, HER2, etc.), andparticularly tumor and patient specific neoantigens (i.e., neoepitopes).As used herein, the tumor-associated antigen refers any antigen that canbe presented on the surface of the tumor cells, which includes aninflammation-associated peptide antigen, a tumor associated peptideantigen, a tumor specific peptide antigen, and a cancer neoepitope.

In some embodiments, the tumor associated antigen is a tumor-specific,patient-specific neoepitope. It is contemplated that patient- andtumor-specific neoantigens or neoepitopes can be identified viaanalyzing and comparing omics data from diseased tissue and healthytissue of a patient, (e.g., via whole genome sequencing and/or exomesequencing, etc.) as is described in US20120059670 and US20120066001.For example, the tumor associated antigens and neoepitopes (which aretypically patient-specific and tumor-specific) can be identified fromthe omics data obtained from the cancer tissue of the patient or normaltissue (of the patient or a healthy individual), respectively. Omicsdata of tumor and/or normal cells preferably comprise a genomic data setthat includes genomic sequence information. Most typically, the genomicsequence information comprises DNA sequence information that is obtainedfrom the patient (e.g., via tumor biopsy), most preferably from thetumor tissue (diseased tissue) and matched healthy tissue of the patientor a healthy individual. For example, the DNA sequence information canbe obtained from a pancreatic cancer cell in the patient’s pancreas(and/or nearby areas for metastasized cells), and a normal pancreaticcells (non-cancerous cells) of the patient or a normal pancreatic cellsfrom a healthy individual other than the patient.

In one especially preferred aspect of the inventive subject matter, DNAanalysis is performed by whole genome sequencing and/or exome sequencing(typically at a coverage depth of at least 10x, more typically at least20x) of both tumor and matched normal sample. Alternatively, DNA datamay also be provided from an already established sequence record (e.g.,SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequencedetermination. Therefore, data sets may include unprocessed or processeddata sets, and exemplary data sets include those having BAM format, SAMformat, FASTQ format, or FASTA format. However, it is especiallypreferred that the data sets are provided in BAM format or as BAMBAMdiff objects (see e.g., US2012/0059670A1 and US2012/0066001A1).Moreover, it should be noted that the data sets are reflective of atumor and a matched normal sample of the same patient to so obtainpatient and tumor specific information. Thus, genetic germ linealterations not giving rise to the tumor (e.g., silent mutation, SNP,etc.) can be excluded such that the neoepitope is filtered against knownhuman SNP and somatic variations. Of course, it should be recognizedthat the tumor sample may be from an initial tumor, from the tumor uponstart of treatment, from a recurrent tumor or metastatic site, etc. Inmost cases, the matched normal sample of the patient may be blood, ornon-diseased tissue from the same tissue type as the tumor.

The so obtained neoepitopes may then be subject to further detailedanalysis and filtering using predefined structural and expressionparameters, and sub-cellular location parameters. For example, it shouldbe appreciated that neoepitope sequences are only retained provided theywill meet a predefined expression threshold (e.g., at least 20%, 30%,40%, 50%, or higher expression as compared to normal) and are identifiedas having a membrane associated location (e.g., are located at theoutside of a cell membrane of a cell). Further contemplated analyseswill include structural calculations that delineate whether or not aneoepitope or a tumor associated antigen, or a self-lipid is likely tobe solvent exposed, presents a structurally stable epitope, etc. Furtherdetails on identification of patient-specific neoantigens and/orcancer-specific, patient-specific neoantigens are described in detail inthe international patent application No. PCT/US16/56550.

Moreover, it is especially contemplated that the tumor-related antigenis a high-affinity binder to at least one MHC Class I sub-type or atleast one MHC Class II sub-type of an HLA-type of the patient, which maybe determined in silico using a de Bruijn graph approach as, forexample, described in WO 2017/035392, or using conventional methods(e.g., antibody-based) known in the art. The binding affinity of thehuman disease-related antigen is tested in silico to the determinedHLA-type. The preferred binding affinity can be measured by lowest KD,for example, less than 500 nM, or less than 250 nM, or less than 150 nM,or less than 50 nM, for example, using NetMHC. Most typically, theHLA-type determination includes at least three MHC-I sub-types (e.g.,HLA-A, HLA-B, HLA-C, etc.) and at least three MHC-II sub-types (e.g.,HLA-DP, HLA-DQ, HLA-DR, etc.), preferably with each subtype beingdetermined to at least 4-digit depth. It should be appreciated that suchapproach will not only identify specific neoantigens that are genuine tothe patient and tumor, but also those neoantigens that are most likelyto be presented on a cell and as such most likely to elicit an immuneresponse with therapeutic effect.

Of course, it should be appreciated that matching of the patient’sHLA-type to the patient- and cancer-specific neoantigen can be doneusing systems other than NetMHC, and suitable systems include NetMHC II,NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep,PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J ImmunolMethods 2011;374:1-4). In calculating the highest affinity, it should benoted that the collection of neoantigen sequences in which the positionof the altered amino acid is moved (supra) can be used. Alternatively,or additionally, modifications to the neoantigens may be implemented byadding N- and/or C-terminal modifications to further increase binding ofthe expressed neoantigen to the patient’s HLA-type. Thus, neoantigensmay be native as identified or further modified to better match aparticular HLA-type.

Moreover, where desired, binding of corresponding wild type sequences(i.e., neoantigen sequence without amino acid change) can be calculatedto ensure high differential affinities. For example, especiallypreferred high differential affinities in MHC binding between theneoantigen and its corresponding wild type sequence are at least 2-fold,at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold,at least 1000-fold, etc.

In addition, the omics information (especially where the omicsinformation comprises whole genome sequencing or exome sequencing, RNAsequence and transcription data, and (preferably quantitative)proteomics information) can also be used to determine the status ofvarious cell signaling pathways. Such pathway information, andespecially in conjunction with mutational information, may revealfurther druggable targets within a cell that are independent fromanatomical features of the tumor (e.g., presence of HER2 signaling in anon-breast cancer). Particularly preferred pathway analyses that arebased on omics information include those described in WO 2011/139345, WO2013/062505, WO 2014/193982, WO 2014/059036, WO 2014/210611, WO2015/184439, and WO 2016/118527. Viewed from a different perspective,omics data in contemplated treatments and uses will be employed to both,inform generation of immune therapeutic compositions as well as informselection of chemotherapeutic drugs based on pathway information ratherthan tumor type and location. Therefore, suitable omics data includewhole genome sequencing data, exome sequencing data, RNA sequence andtranscription data, and proteomics data (e.g., quantitative proteomicsdata from mass spectroscopic analyses).

In some embodiments, the tumor associated antigen or neoepitopes can bea polytope. As used herein, a polytope refers a tandem array of two ormore antigens (or neoepitopes) expressed as a single polypeptide.Preferably, two or more human disease-related antigens are separated bya linker or spacer peptides. Any suitable length and order of peptidesequence for the linker or the spacer can be used. However, it ispreferred that the length of the linker peptide is between 3-30 aminoacids, preferably between 5-20 amino acids, more preferably between 5-15amino acids. Also inventors contemplates that glycine-rich sequences(e.g., gly-gly-ser-gly-gly, etc.) are preferred to provide flexibilityof the polytope between two antigens.

The notable reduction in potentially useful neoepitopes as describedabove is exemplarily shown in FIG. 1 where a patients tumor tissue iscompared using the same patient’s normal tissue using omics analysis andneoepitope discovery. Once identified, an adenovirus is then geneticallyengineered to include an expression cassette that includes nucleic acidsegments that encode one or more neoepitopes of the patient. Suchmodified virus can then be used as a recombinant viral vaccine. FIG. 2exemplarily illustrates a selection process in which all neoepitopesidentified by omics analysis (left bar) are first filtered by expressionlevel. Here, non-expressed neoepitopes are eliminated, resulting in asubstantial reduction in potential neoepitopes (middle bar). Thesefiltered neoepitopes are further filtered against the patients MHC type,and as can be seen from the Figure (right bar), the number ofneoepitopes that can be presented by the patient’s antigen presentingcells is once more dramatically reduced. The neoepitopes can then beused in therapy for example in a recombinant virus/recombinantexpression system.

In especially contemplated aspects, a nucleic acid sequence encoding oneor more tumor-associated antigen(s) and/or neoepitopes can be placed inan expression vector. The recombinant nucleic acid is then inserted intoan expression vector such that the nucleic acid can be delivered to anantigen presenting cell (e.g., dendritic cells, etc.) of the patient, orto transcribe the nucleic acid sequence in bacteria or yeast so that therecombinant protein encoded by the nucleic acid sequence can be, as awhole, or as fragments, delivered to the antigen presenting cell. Anysuitable expression vectors that can be used to express protein arecontemplated. Especially preferred expression vectors may include thosethat can carry a cassette size of at least 1 k, preferably 2 k, morepreferably 5 k base pairs.

Thus, in one embodiment, a preferred expression vector includes a viralvector (e.g., non-replicating recombinant adenovirus genome, optionallywith a deleted or non-functional E1 and/or E2b gene). Where theexpression vector is viral vector (e.g., an adenovirus, and especiallyAdV with E1 and E2b deleted), it is contemplated that the recombinantviruses including the recombinant nucleic acid may then be individuallyor in combination used as a therapeutic vaccine in a pharmaceuticalcomposition, typically formulated as a sterile injectable compositionwith a virus titer of between 10⁶-10¹³ virus particles, and moretypically between 10⁹-10¹² virus particles per dosage unit. For example,the virus vaccine can be formulated with a virus titer at least 10⁶virus particles/day, or at least 10⁸ virus particles/day, or at least10¹⁰ virus particles/day, or at least 10¹¹ virus particles/day.Alternatively, virus may be employed to infect patient (or other HLAmatched) cells ex vivo and the so infected cells are then transfused tothe patient.

In still further embodiments, the expression vector can also be abacterial vector that can be expressed in a genetically engineeredbacterium, which expresses endotoxins at a level low enough not to causean endotoxic response in human cells and/or insufficient to induce aCD-14 mediated sepsis when introduced to the human body. One exemplarybacteria strain with modified lipopolysaccharides includes ClearColi®BL21 (DE3) electrocompetent cells. This bacteria strain is BL21 with agenotype F- ompT hsdSB (rB- mB-) gal dcm lon λ(DE3 [laI lacUV5-T7 gene 1ind1 sam7 nin5]) msbA148 ΔgutQΔkdsD ΔlpxLΔlpxMΔpagPΔlpxPΔeptA. In thiscontext, it should be appreciated that several specific deletionmutations (ΔgutQ ΔkdsD ΔlpxL ΔlpxMΔpagPΔlpxPΔeptA) encode themodification of LPS to Lipid IVA, while one additional compensatingmutation (msbA148) enables the cells to maintain viability in thepresence of the LPS precursor lipid IVA. These mutations result in thedeletion of the oligosaccharide chain from the LPS. More specifically,two of the six acyl chains are deleted. The six acyl chains of the LPSare the trigger which is recognized by the Toll-like receptor 4 (TLR4)in complex with myeloid differentiation factor 2 (MD-2), causingactivation of NF-ƙB and production of proinflammatory cytokines. LipidIVA, which contains only four acyl chains, is not recognized by TLR4 andthus does not trigger the endotoxic response. While electrocompetentBL21 bacteria is provided as an example, the inventors contemplates thatthe genetically modified bacteria can be also chemically competentbacteria. Alternatively, or additionally, the expression vector can alsobe a yeast vector that can be expressed in yeast, preferably, inSaccharomyces cerevisiae (e.g., GI-400 series recombinantimmunotherapeutic yeast strains, etc.). Of course, it should beappreciated that recombinant nucleic acids contemplated herein need notbe limited to viral, yeast, or bacterial expression vectors, but mayalso include DNA vaccine vectors, linearized DNA, and mRNA, all of whichcan be transfected into suitable cells following protocols well known inthe art.

Moreover, it should be recognized that the vaccine composition maychange over the course of the vaccination regimen, in terms offormulation and in terms of antigen composition. For example, a primevaccination may be performed using a recombinant bacterial or yeastexpression system as described above, which is then followed in theboost phase using with an adenoviral vaccination. Additionally, thevaccination regimen may also use different routes of administration,including subcutaneous or intramuscular administration of a bacterial oryeast vaccine composition, and an intratumoral or intravenousadministration of an adenoviral vaccine composition. Similarly, in lesspreferred aspects, the prime vaccine composition may include both tumorassociated antigens and neoepitopes, while the boost vaccination mayinclude or encode only neoepitopes as the immunogenic agent.

Still further, it is contemplated that the vaccine regimen may usevarious timelines for administration. However, it is generallycontemplated that the timing of prime/boost will follow an anticipatedtiming of immune response with respect to antigen processing andpresentation and T cell stimulation and proliferation. Therefore, thevaccine compositions will at least initially be administered at least 2days apart, more typically within a window of 3-5 days, or 5-7 days, or7-10 days, or 10-14 days, and in some cases even longer. For subsequentadministrations in the boost phase (e.g., fourth, fifth, etc.administration), the interval may be longer, including several weeks,months, or even years. In some embodiments, the dose of the virusformulation can be gradually increased during the schedule, or graduallydecreased during the schedule. In still other embodiments, severalseries of administration of virus formulation can be separated by aninterval (e.g., one administration each for 3 consecutive days and oneadministration each for another 3 consecutive days with an interval of 7days, etc.).

With respect to contemplated immune stimulation regimens, it isgenerally preferred that such regimen comprises at least a first and asecond administration of one or more immune stimulatory compositions,and it is generally preferred that the immune stimulatory compositioncomprises an immune stimulatory cytokine. For example, especiallypreferred cytokines include various interleukins, and particularly IL-2,IL-12, IL-15, IL-17, IL-21, as well as other cytokines including Th1cytokines (e.g., IFN-γ, TNFα, etc.). Suitable dosages for the immunestimulation will typically be within the range of clinically acceptabledosages, which art well known in the art. Moreover, it is furthercontemplated that the immune stimulatory compositions may also includemodified forms of the above noted proteins, and especially modifiedforms include those that increase serum half life time and/or reducetoxicity. For example, contemplated immune stimulating compositions mayinclude truncated IL-2 that lacks the PEP portion, or ALT-803, which isan IL-15 based chimeric protein complex with IL-15 superagonistfunction. Still further contemplated immune stimulatory compounds mayalso be target specific, for example, by coupling to an antibody orportion thereof, wherein the antibody typically binds a tumor epitope, aneoepitope, or a component of a necrotic cell (e.g., dsDNA, histones,etc.). Once more, it should be noted that suitable dosages for theimmune stimulation using such compounds will typically be within therange of clinically acceptable dosages, which art well known in the art.

Alternatively, or additionally, further contemplated immune stimulatorycompounds include various TLR ligands and/or NOD ligands that enhance animmune response. For example, suitable TLR ligands include TLR2, TLR3,TLR4, TLR7, or TLR8, and especially include mRNA, dsDNA, ssRNA, CpG,glycolipids, etc. In still further contemplated aspects, immunestimulatory compounds may also include an OX40 agonistic antibody. Thus,numerous immune stimulatory compounds are deemed suitable, and allreasonable combinations thereof.

Of course, it should be recognized that the immune stimulatory compoundsmay change over the course of the treatment regimen, both in terms oftype and the dosage of the immune stimulatory compounds. For example,initial stimulation maybe may be performed using more acute effectcompounds (e.g., IL-2, IL-15), while subsequent administrations mayemploy immune stimulatory compounds with longer lasting effects (e.g.,ALT-803). On the other hand, immune stimulatory compounds may also beadministered in a target specific manner as noted above to reducesystemic (toxic) effects. The immune stimulatory regimen may usedifferent routes of administration, including subcutaneous orintramuscular administration, however, intratumoral or intravenousadministration are particularly preferred.

Still further, it is contemplated that the immune stimulatory regimenmay use various timelines for administration. However, it is generallycontemplated that the first administration will be after the primeadministration of the vaccine as already noted above. Subsequentadministrations will generally be in relatively rapid succession toelicit a substantially continuous stimulatory effect. Therefore, theimmune stimulatory compounds will be administered about 1 day apart,more typically within a window of 1-2 days, or 2-3 days, or 1-3 days, or2-4 days, and in some cases even longer intervals. For still lateradministrations (e.g., after the vaccine boost phase), the interval maybe longer, including several weeks, months, or even years.

It is still further generally preferred that the immune stimulatorycompounds are at least administered within the prime/boost vaccineregimen, and that the immune stimulatory regimen overlaps to at leastsome degree with a checkpoint inhibition regimen as described in moredetail below. While not limiting to the inventive subject matter, it isbelieved that upon administration of the prime vaccination, an immunestimulatory phase that supports immune cell activation and a regimenthat reduces checkpoint inhibition will modulate the immune system toprovide an optimum response to the prime and contemporaneous/subsequentboost phase.

Therefore, it is generally preferred that the checkpoint inhibitionregimen comprises at least a first and a second administration of acheckpoint inhibitor, and all known checkpoint inhibitors are deemedsuitable for use herein. For example, suitable checkpoint inhibitorsinclude those that interfere with signaling through PD-1 (e.g.,pembrozilumab, nivolumab), PD-L1 (e.g., atezolizumab, avelumab,durvalumab), and/or CTLA4 (e.g., ipilimumab). There are numerous suchinhibitors known in the art and all of those are deemed suitable for useherein. Suitable dosages for the immune stimulation will typically bewithin the range of clinically acceptable dosages, which art well knownin the art. Likewise, where a tumor secrets soluble ligands tocheckpoint receptors, it is contemplated that antibodies scavenging suchsoluble factors may be used to reduce adverse effects of such solubleforms.

As noted above, it is contemplated that the checkpoint inhibitionregimen may employ various timelines for administration. However, it isgenerally contemplated that the first administration will be after thefirst administration of the vaccine and also after the firstadministration of the immune stimulatory compound. Thus, the immunestimulation and checkpoint inhibition will at least partially overlap.Subsequent administrations will generally be in relatively rapidsuccession to elicit a substantially continuous stimulatory effect.Therefore, the checkpoint inhibitors will be administered about 1 dayapart, more typically within a window of 1-2 days, or 2-3 days, or 1-3days, or 2-4 days, and in some cases even longer intervals. For stilllater administrations (e.g., after the vaccine boost phase), theinterval may be longer, including several weeks, months, or even years.

With respect to the administration of the compound that biases theimmune response towards a Th1 response and/or maintains an immuneresponse against the tumor antigen with development of immune memory, itis generally preferred that the compound is a cytokine, and especiallyIL-12. Most preferably, to increase the site specificity and serumhalf-life of the IL-12, it is contemplated that the IL-12 is conjugatedto an antibody or portion thereof, wherein the antibody binds to a tumorantigen, a tumor neoepitope, or a component of a necrotic cell. Thus,especially preferred compounds include NHS IL-12 and derivativesthereof. Alternatively, various other compounds can be used, includingplant based compositions (e.g., PLoS Negl Trop Dis. 2015 Jan; 9(1):e3321), orally delivered deoxunucleotides (e.g., Molecular Therapy,Vol.23, Issue 2, 222 - 223), ribavirin (e.g., PLoS One. 2012; 7(7):e42094.), and less preferably IL-4 and IL-10.

Administration of such compounds will generally follow dosages wellknown in the art, and all such dosages are deemed suitable for useherein. As noted above, it is contemplated that the Th1stimulation/immune support may employ various timelines foradministration. However, it is generally contemplated that the firstadministration will be after the second or subsequent administration ofthe vaccine and also after the administrations of the immune stimulatorycompound and checkpoint inhibitor. Thus, the Th1 stimulation willtypically coincide with the boost phase of vaccination, and mosttypically provide a support function that maintains the immunestimulation. Subsequent administrations will generally be in relativelyslow succession to maintain a continuous stimulatory effect. Therefore,the compounds that stimulate Th1 will be administered, where desired,about 3 day apart, more typically within a window of 2-5 days, or 5-7days, or 7-14, and in some cases even longer intervals. For still lateradministrations (e.g., with further vaccine boost administrations), theinterval may be longer, including several weeks, months, or even years.

Of course, it should be appreciated that contemplated treatments mayfurther include other therapeutic compositions that complement orotherwise support the methods described herein. For example,contemplated methods may include administration of autologous orheterologous NK cells to a patient, and particularly NK cells that aregenetically modified to exhibit less inhibition. For example, thegenetically modified NK cell may be a NK-92 derivative that is modifiedto have a reduced or abolished expression of at least one killer cellimmunoglobulin-like receptor (KIR), which will render such cellsconstitutively activated. Of course, it should be noted that one or moreKIRs may be deleted or that their expression may be suppressed (e.g.,via miRNA, siRNA, etc.), including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4,KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5,KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells may beprepared using protocols well known in the art. Alternatively, suchcells may also be commercially obtained from NantKwest as aNK cells(‘activated natural killer cells). Such cells may then be furthermodified to express the co-stimulatory molecules as further discussedbelow. In addition, contemplated NK cells suitable for use herein alsoinclude those that have abolished or silenced expression of NKG2A, whichis an activating signal to Tregs and MDSCs.

Alternatively, the genetically engineered NK cell may also be an NK-92derivative that is modified to express a high-affinity Fcγ receptor(CD16-158V). Sequences for high-affinity variants of the Fcγ receptorare well known in the art, and all manners of generating and expressionare deemed suitable for use herein. Expression of such receptor isbelieved to allow specific targeting of tumor cells using antibodiesproduced by the patient in response to the treatment contemplatedherein, or supplied as therapeutic antibodies, where those antibodiesare specific to a patient’s tumor cells (e.g., neoepitopes), aparticular tumor type (e.g., her2neu, PSA, PSMA, etc.), or antigensassociated with cancer (e.g., CEA-CAM). Advantageously, such cells maybe commercially obtained from NantKwest as haNK cells (‘high-affinitynatural killer cells) and may then be further modified (e.g., to expressco-stimulatory molecules).

In further aspects, genetically engineered NK cells may also begenetically engineered to express a chimeric T cell receptor. Inespecially preferred aspects, the chimeric T cell receptor will have anscFv portion or other ectodomain with binding specificity against atumor associated antigen, a tumor specific antigen, and/or a neoepitopeof the patient as determined by the omics analysis. As before, suchcells may be commercially obtained from NantKwest as taNK cells(‘target-activated natural killer cells’) and further modified asdesired. Where the cells have a chimeric T cell receptor engineered tohave affinity towards a cancer associated antigen or neoepitope, it iscontemplated that all known cancer associated antigens and neoepitopesare considered appropriate for use. For example, tumor associatedantigens include CEA, MUC-1, CYPB1, PSA, Her-2, PSA, brachyury, etc.

Moreover, it should be noted that the methods and uses contemplatedherein also include cell based treatments with cells other than (or inaddition to) NK cells. For example, suitable cell based treatmentsinclude T cell based treatments. Among other options, it is contemplatedthat one or more features associated with T cells (e.g., CD4+ T cells,CD8+ T cells, etc.) can be detected. More specifically, contemplatedomics analysis can identify specific neoepitopes (e.g., 8-mers to12-mers for MHC I, 12-mers to 25-mers for MHC II, etc.) that can be usedfor the identification of neoepitope reactive T cells bearing a specificT cell receptor against the neoepitopes/MHC protein complexes. Thus, themethod can include harvesting the neoepitope reactive T cells. Theharvested T cells can be grown or expanded (or reactivated whereexhausted) ex vivo in preparation for reintroduction to the patient.Alternatively, the T cell receptor genes in the harvested T cells can beisolated and transferred into viruses, or other adoptive cell therapiessystems (e.g., CAR-T, CAR-TANK, etc.). Beyond neoepitopes, the omicsanalyses can also provide one or more tumor associated antigens (TAAs).Therefore, one can also harvest T cells that have receptors that aresensitive to the TAAs identified from these analyses. These cells can begrown or cultured ex vivo and used in a similar therapeutic manner asdiscussed above. The T cells can be identified by producing syntheticversions of the peptides and bind them with commercially produced MHC orMHC-like proteins, then using these ex vivo complexes to bind to thetarget T cells. One should appreciated that the harvested T cells canincluded T cells that have been activated by the patient’s immuneresponse to the disease, exhausted T cells, or other T cells that areresponsive to the discussed features.

EXAMPLES

The following description provides exemplary protocols to treat cancerin a patient according to the inventive subject matter. It should beunderstood that while these protocols list specific compounds andcompositions alone or in combination, various alternative compounds andcompositions may be provided with the same or similar effect. Moreover,dosage and schedules may change according to patient age, stage ofcancer, and overall health condition.

Example 1

As can be readily seen from FIG. 3 , the timed sequence of prime/boostvaccination with immune stimulation and checkpoint inhibition and Th1stimulation had dramatic effect (tumor volumes were significantlyreduced after 40 days of combination treatment) as compared to anytreatment regimen that lacked one of the components. This remarkablepicture is also reflected in the histopathological analysis shown inFIG. 4 . Here, photomicrographs are shown with CD8+ tumor infiltratingcells stained. Once more, the timed sequence of prime/boost vaccinationwith immune stimulation, checkpoint inhibition, and Th1 stimulationresulted in a significant increase in T cells infiltrating the tumor.FIG. 5 shows the results for a follow-up study indicating days after NHSIL-12 administration. Again, the effect of the timed sequence ofprime/boost vaccination with immune stimulation and checkpointinhibition and Th1 stimulation is readily apparent and resulted in allbut two cured animals.

Example 2

Cell culture: MC38 and RMA-S cells were grown in RPMI-1640 withL-glutamine (Corning) supplemented with 10% (v/v) fetal bovine serum(Atlanta Biologicals) and 1% (v/v) antibiotic/antimitotic solution(Corning). All cells were cultured at 37° C., 5% CO2.

Animals and tumor implantation: Mice were handled in accordance with theAssociation for Assessment and Accreditation of Laboratory Animal Careguidelines, and under the approval of the NIH Intramural Animal Care andUse Committee. Mice were bred and housed at the NIH. Tumors were inducedby implanting 3×105 tumor cells subcutaneously. All studies utilizedfemale C57BL/6 animals.

Identification of tumor variants and neoepitopes: Single nucleotidevariants (SNVs) and insertions/deletions (indels) were identified aspreviously described (26). Neoepitopes were identified by creating allpossible permutations of 9-mer amino acid sequences derived from anidentified non-silent SNV or indel. Neoepitopes were ranked by RNAexpression as well as allele frequency of the observed coding variant tooffset issues arising from tumor heterogeneity. NetMHC 3.4(http://www.cbs.dtu.dk/services/NetMHC-3.4/) (27,28) was used to predictneoepitope binding to a specific MHC H-2 allele. Neoepitopes withpredicted binding affinities < 500 nM were retained for furtheranalysis.

Peptide synthesis: Peptides were synthesized by Bio-Synthesis orGenScript to >85% purity.

Cell isolation and preparation: Spleens were harvested, dissociatedthrough 70 □m filters and subjected to ACK lysis to obtain splenocytesfor analysis. Tumors were harvested, cut into small pieces and incubatedfor 1 hour in a digestion cocktail comprised of RPMI supplemented with5% (v/v) FBS, 2 mg/mL Collagenase Type I (Worthington BiochemicalCorporation) and 40 U/mL DNase I (Calbiochem). Following digestion,tumors were ground through 70 µm filters and tumor- infiltratingleukocytes (TILs) were enriched using a 40%/70% Percoll (Sigma)gradient. Peripheral blood mononuclear cells (PBMCs) were isolated fromwhole, anticoagulated mouse blood by layering over lymphocyte separationmedium (MP Biomedicals) and collecting lymphocyte layer.

Flow cytometric assays: All antibodies used for flow cytometric analysisare fully described in Supplementary Table S1. Peptide binding assayswere performed using RMA-S cells incubated with individual peptides at50 µg/mL overnight. Following incubation, cells were stained withanti-MHC antibodies. Data were acquired using a FACS Calibur (BDBiosciences), and reported as an in vitro binding score, which is thepercentage of RMA-S cells expressing MHC on their surface.Tumor-infiltrating leukocytes were stained for immune cell subsets anddata were collected using an Attune NxT flow cytometer (ThermoFisher) orBD LSRFortessa (BD Biosciences). Cell populations were identified asfollows: CD8+ T cells: live/CD45+/CD3+/CD8+; macrophages:live/CD45+/CD3-/CD11b+F4/80+. For intracellular cytokine staining,splenocytes were cultured with indicated peptides for 4 hours, at whichtime GolgiPlug/GolgiStop (BD Biosciences) were added. Cultures wereincubated an additional 20 hours, and fixed, permeabilized and stainedfor TNF and IFNγ production. Data were collected using an Attune NxTflow cytometer.

Vaccination and treatment with immune-modulators: Animals werevaccinated with pools of 9-mer or 25-mer neoepitope peptides (100 µgeach peptide), emulsified in Montanide ISA 51 VG (Seppic), administeredsubcutaneously. Adenoviral vectors encoding TWIST1 or neoepitopes werekindly produced and provided by the Etubics/NantCell Corporation. 1010viral particles encoding the multi-epitope virus were administeredsubcutaneously. The admixed virus was administered by injecting animalswith 1010 viral particles each of which is a single-neoepitope encodingviruses subcutaneously. N-803 was kindly provided by Nantworks, and 1 µgwas administered subcutaneously into animals. Anti-PD-L1 (10F.9G2,BioXCell, 200 µg) was administered intraperitoneally. Murine NHS-IL12was administered at a dose of 50 µg.

Assessment of immunity: Splenocytes or PBMCs were harvested and ex-vivoantigen-dependent cytokine secretion was assessed using IFNγ (BDBioscience) or TNFα (Cellular Technology Ltd.) ELISPOT. Target peptides(10 µg/mL final concentration) were incubated with cells overnight.Assays were performed according to manufacturers’ instructions. ELISPOTdata are adjusted to the number of spots/million splenocytes aftersubtracting the number of spots in paired wells containing a controlpeptide.

Immunohistochemistry: Tumors, fixed in Z-fix (Anatech), were paraffinembedded and sectioned. Slides were stained for CD8a (4SM16) using theOpal Multiplex Immunohistochemistry Kits (PerkinElmer). Images wereacquired using an Axio Scan.Z1 slide scanner (Zeiss).

Depletion studies: Depletions were monitored in the peripheral blood viaflow cytometry.

NanoString and T cell receptor (TCR) sequencing: Isolatedtumor-infiltrating leukocytes were enriched using a CD45+ or CD4+/CD8+murine TIL microbead kit (Miltenyi Biotech). RNA was purified from CD45+cells using the RNeasy mini kit (Qiagen). Gene expression was assayedusing the murine nCounter PanCancer Immune Profiling Panel (NanoString).Nanostring data was normalized using the nSolver Analysis Software 4.0.Genes whose expression was altered by at least 2-fold in biologicalreplicates compared to non-treated control animals were considered to besignificant. TCR diversity was assessed using genomic DNA purified fromtumor-infiltrating CD4+ and CD8+ cells using the QIAamp DNA mini kit(Qiagen). TCRβ chain sequencing was performed by AdaptiveBiotechnologies and analyzed using the Immunoseq analyzer. The top 100TCR sequences were analyzed.

Data and statistical analysis: Flow cytometry data were analyzed usingFlowJo (v10, BD Biosciences). All hierarchical clusters were generatedusing the Partek Genomics Suite. Statistics were performed usingGraphPad Prism (v7; GraphPad Software). All data points represent themean ± SEM and P ≤0.05 was considered significant. Significance isindicated within figures as follows: ^(∗) P ≤ 0.05, ^(∗∗) P ≤ 0.01,^(∗∗∗) P ≤ 0.001, ^(∗∗∗∗) P ≤ 0.0001.

Whole-exome DNA and RNA sequencing was performed on two MC38 tumors, andexpressed nonsynonymous mutations were identified using a tumor-normalDNA analysis from splenocytes harvested from the same animals (FIG. 6A).Although both tumors were induced using MC38 cells harvested from thesame culture flasks, the inventors observed a large difference in numberof DNA mutations, and potential neoepitopes identified in each of thetumors analyzed (Table 1).

TABLE 1 Tumor 1 Tumor 2 Number of nonsynonymous mutations 16828 8676Number of expressed nonsynonymous mutations 7098 (42.2%) 3548 (40.9%)Number of nonsynonymous mutations predicted to bind MHC 124 (0.7%) 66(0.8%) Number of shared neoepitopes 51 (0.3-0.5%)

This observation is supportive of previous studies in which the MC38tumor cell line has been identified as being microsatellite-unstable. Intotal, the inventors identified 51 potential neoepitopes, representing43 unique nonsynonymous mutations, shared among both tumors assayed(Table 1). There continues to be no defined method of selectingappropriate nonsynonymous mutations to target with a vaccine; theinventors assumed that an ideal neoepitope would be both highlyexpressed and have a high affinity for binding MHC. Based upon thismetric, the inventors initially ranked potential neoepitopes by dividingthe relative expression by predicted MHC binding affinity. In addition,the inventors also utilized a second metric, which ranked neoepitopessolely by their predicted MHC binding affinity. Thirteen peptides,representing the top 10 peptides from both metrics were synthesized.Seven of these 13 neoepitopes were capable of binding MHC in-vitro (FIG.6B), and the immunogenicity of each peptide was assessed by vaccinatingnon-tumor bearing mice with pools of neoepitope peptides emulsified inMontanide ISA 51 VG adjuvant (Montanide). Using an ex-vivo IFNγ ELISPOTassay, 6/13 of the neoepitopes were found to induce immune responses inrepeated experiments using non-tumor bearing animals (FIG. 6C, reactivepeptides highlighted with shaded bars). Based upon these observations,we chose to utilize a vaccination strategy incorporating a pool of four9-mer neoepitope peptides (Jak1, Olfr99, Ptgfr and Trp53) emulsified inMontanide. When administered as a single agent, the neoepitope vaccineinduced a low level of immunity, but failed to provide any survivalbenefit as compared to control animals. In an effort to enhance theeffectiveness of the neoepitope vaccine, the inventors combined it withthe IL15 superagonist fusion protein N-803 and anti-PD-L1 MAb. Using thetreatment regimen outlined in FIG. 6D, N-803 and anti-PD-L1 synergizedto increase the immunogenicity of the 9-mer, but not 25-mer neoepitopevaccines in tumor bearing animals (FIGS. 6E and 6F). This enhancement inimmunogenicity seen when combining our 9-mer neoepitope vaccine withN-803 and anti-PD-L1 correlated with only a slight, but significantincrease in the survival of treated, as compared to control, animals(FIG. 6G). This survival benefit was seen only in animals mounting arobust immune response against vaccine components, as assessed byperforming an ELISPOT using peripheral blood collected from animals onday 13 of tumor growth (FIG. 6H).

Using the treatment regimen depicted in FIG. 7A, the inventors assayedthe immune response in vaccinated animals on days 11, 18 and 25 of tumorgrowth, and observed that the magnitude of the immunity generatedagainst vaccine components decreased over time. Interestingly, theinventors observed that the neoepitope vaccine also resulted in thein-situ expansion of T cells reactive against neoepitopes not containedin the vaccine. However, the magnitude of these de-novo immune responsesalso diminished over time despite continued vaccination (FIG. 7B). In aneffort to promote the maintenance of neoepitope-reactive cells withinthe tumor microenvironment, the inventors incorporated a singleinjection of NHS-IL12 to the vaccine regimen on day 18 of tumor growth.Animals treated with the combination of anti-PD-L1, NHS-IL12, N-803 andneoepitope vaccine were able to maintain a robust immune responseagainst neoepitopes Ptgfr and Trp53, both of which are components of theneoepitope vaccine, along with additional neoepitopes expressed by MC38tumors, but not included in the vaccine (FIG. 7B). Interestingly,treatment of mice with N-803, anti-PD-L1 and NHS-IL12, but withoutvaccine, promoted only the expansion of T cells specific for a peptide(P15e) derived from GP70, an endogenous retrovirus protein expressed byMC38 cells, but not to any of the neoepitopes assayed (FIG. 7B).

In the presence or absence of vaccination, animals treated with N-803and anti-PD-L1 showed no evidence of tumor control (FIG. 7C); inaddition, animals treated with the combination of N-803, anti-PD-L1 andNHS-IL12, and no vaccine, had transient tumor control (FIG. 7C), withthe median overall survival increasing from 21 days in non-treatedanimals to 39 days in animals treated with the triple combination. Theaddition of a neoepitope vaccine to this combination, however resultedin a dramatic regression of the majority of MC38 tumors (FIG. 7C, rightpanel). There was also a correlation as to which animals responded tothis treatment via assessing the magnitude of the immune responsegenerated against vaccine components as measured using peripheral bloodcollected from animals on day 13 of tumor growth, prior to theadministration of NHS-IL12 (FIG. 7D). Animals whose tumors resolvedfollowing treatment remained tumor- free after the cessation ofvaccination on day 39, and 4/6 animals subsequently resisted tumorrechallenge with MC38 on day 76. These re-challenged animals likely wereable to completely clear any residual MC38 cells, as the inventors didnot observe tumor outgrowth, even after depleting animals of CD8+ Tcells 81 days after the second tumor implantation (FIG. 7E).Interestingly, the inventors observed a higher proportion of T cellsproducing TNFα in response to cascade neoepitopes as compared to thosecontained within the vaccine as assessed by ELISPOT (FIG. 7F).Similarly, flow cytometric analysis demonstrated a larger predominanceof poly-functional T cells producing antigen-dependent IFNγ and TNFα inT cells reactive against cascade neoepitopes as compared to thosecontained within the vaccine (FIG. 7G). Animals treated with vaccine andNHS-IL12, using the same treatment timeline outlined in FIG. 7A, wereable to mediate some degree of tumor regression in the absence of N-803and anti-PD-L1 treatment; however, all of the treated animals eventuallysuccumbed to progressive tumor growth. (FIG. 7H)

The dramatic regression of tumors in the combinatorial treatment groupwas associated with limited toxicity. All tissues examined werehistologically normal with the exception of focal areas of glandularepithelial necrosis without inflammation in the duodenum, andhypercellularity in the small bowel of a treated animal. There was alsoa transient, slight increase in serum liver enzyme levels in treatedmice that was not associated with any liver pathology.

To ascertain if a vaccine consisting of a single neoepitope was capableof mediating tumor regression, tumor-bearing animals were treated witheither a single 9-mer or a pool of all four neoepitope peptides incombination with N-803, anti-PD-L1 and NHS-IL12. The pool of neoepitopeswas more efficient at inducing the regression of MC38 tumors than any ofthe single peptide vaccinations (FIG. 8A). To determine the cellpopulations mediating effective anti-tumor immunity, animals weredepleted of NK 1.1+, CD4+ or CD8+ T cells either beginning on day 1 oftumor growth (early depletion) or day 15 of tumor growth (latedepletion). Animals depleted of NK1.1+ cells early during tumor growthwere able to resolve MC38 tumors with kinetics similar to those seen innon-depleted animals. Early depletion of CD4+ cells in treated animalswas associated with a more rapid regression of tumors as compared tonon-depleted animals. Late depletion of NK 1.1+ cells was associatedwith only 2/10 animals controlling tumor growth. Late depletion of CD4+cells was associated with a rate of tumor resolution similar to thatseen in non-depleted treated animals. As expected, both early and latedepletion of CD8+ cells were associated with a lack of response totreatment, resulting in progressive tumor growth (FIG. 8B).

Using the same treatment regimen depicted in FIG. 7A, the inventorsassessed which components of the immune system were modulated by each ofthe different agents and their combinations within the tumormicroenvironment. The quadruple treatment regimen was shown to maximallyenhance the infiltration of CD8+ T cells into the tumor (FIGS. 9A and9B). The addition of NHS- IL12 to the treatment regimen had a profoundimpact on both the innate and adaptive immune cells within the tumormicroenvironment. It promoted the expansion of M1 macrophages with acoordinate contraction of M2 macrophages (FIG. 9C); also observed was atrend in the reduction of effector and central memory CD8+ T cells, andan expansion of CD8+ effector memory cells (FIG. 9D). Treatment withvaccine and NHS-IL12 in combination with either N-803 or anti-PD-L1 wasable to induce regression in 60% of MC38 tumors, as compared to 80%observed with the quad-therapy. Protective immunological memory wasgenerated more efficiently in animals treated with either thequad-therapy or vaccine, anti-PD-L1 and NHS-IL12 as compared to animalstreated with N-803, vaccine and NHS-IL12.

To better assess the impact of the treatment regimen on immune cellsinfiltrating the tumor, the inventors performed an analysis of geneexpression in CD45+ cells isolated from day 25 tumors. Treatment of thecombination of N-803, anti-PD-L1 and neoepitope vaccine had littleobservable impact on the expression of immune-related genes as comparedto cells isolated from untreated tumors (FIG. 5A). The addition ofNHS-IL12 to the N-803 and anti-PD-L1 treatment regimen correlated withan increased expression of a large number of genes primarily related toenhancement of the innate immune system. The addition of a neoepitopevaccine to this treatment, which was required to induce maximum tumorclearance, was associated with a great expansion of transcripts relatingto T cell activation and effector functions (FIG. 10A).

To assess the impact of the treatment on the diversity of the immunerepertoire of cells infiltrating the tumor, the inventors sequenced thebeta chain of the T cell receptor (TCRβ) of CD4+ and CD8+ cells isolatedfrom day 25 tumors following treatment with indicated therapies. The useof NHS-IL12, N-803 and anti-PD-L1in the treatment regimen resulted inthe greatest increase in clonality of T cells within the tumor, which ismodestly decreased upon incorporation of the neoepitope vaccine (FIG.10B). To gain a better sense of the clonality of the T cell infiltrateswithin each tumor, the inventors examined the number of clones requiredto make up the top 25% of the productive clones. In mice treated withthe neoepitope vaccine, N-803 and anti-PD-L1, the top 25% of productiverearrangements was composed of 21, 57 and 126 clones. The numberdecreased to 1, 2 and 6 in animals treated with N-803, anti-PD-L1 andNHS-IL12. With the addition of the neoepitope vaccine, which is requiredfor tumor regression, the number of clones were 3, 7 and 22. Theseresults indicate that NHS-IL12 drives the expansion of a limited numberof clones, while the neoepitope vaccine broadens the repertoire, whichassociates with tumor clearance (FIG. 10C). An analysis of the top 100TCRβ sequences detected in each sample revealed that each animal,regardless of treatment, had a unique T cell repertoire (FIG. 10D).

Recombinant adenoviral vectors were produced that encoded either singleneoepitopes or four neoepitopes in a single viral vector (FIG. 11A).Following the treatment schedule outlined in FIG. 11B, tumor-bearinganimals were treated using an admix of four vectors with each singleneoepitope vector administered at spatially separated injection sites,or a single viral vector encoding four neoepitopes (multi-epitope). Boththe admix and multi-epitope vectors resulted in comparable immunitygenerated against the four neoepitopes comprising the vaccine (FIG.11C); however, vaccination with the multi-epitope vector was moreefficient at promoting the in-situ spread of immunity to neoepitopesexpressed by the tumor, but not incorporated into the vector (FIG. 11D).The inventors hypothesized that one could facilitate epitope spreadinduced by the admixed vaccine by mixing the four preparations of viralparticles prior to injection. However, even when all four admixedneoepitopes vaccines are administered together, and presumablyactivating T cells within the same draining lymph nodes, the admixedvaccine continues to be inefficient at mediating epitope spread ascompared to the multi-epitope vaccine. This increased epitope spreadobserved with the administration of the multi-epitope vaccine associatedwith a more efficient tumor resolution in animals vaccinated with themulti- epitope vector, as compared to the admix of single neoepitopevectors (FIG. 11E). This protective anti-tumor immune responsecorrelated with increased tumor infiltration of CD8+ T cells, along witha higher CD8:CD4 T cell ratio (FIG. 11F and G). The inventors did notobserve any differences in the presence of tumor-infiltrating Tregulatory cells among the two experimental groups (FIG. 11H). Animalswhose tumors resolved after treatment remained tumor-free even after thecessation of vaccination on day 39, and all animals subsequentlyresisted subsequent tumor rechallenge.

The inventors sought to determine if the degree of epitope spreaddetected on day 25 of tumor growth associated with a change in the rateof tumor growth in animals treated with the combination of neoepitopevaccine, N-803, anti-PD-L1 and NHS-IL12. The inventors observed apositive association between a decreased rate of tumor growth and thegeneration of epitope spread to numerous neoepitopes. Interestingly,there was no correlation of a decreased rate of tumor growth with themagnitude of immunity generated against the vaccine components or theP15e peptide.

Utilizing the same treatment schedule and regimen including N-803,anti-PD-L1 and NHS-IL12 outlined in FIG. 11B, mice were vaccinated withan adenoviral vector encoding the ‘self’ tumor-associated antigenTWIST1, which is expressed by the MC38 cell line (32), and has been usedin prior anti-cancer vaccine studies (33,34). Tumor regression wasobserved in only 2/10 animals treated with a regimen incorporating theTWIST1-targeted vaccine (FIG. 11I). Interestingly, TWIST1 vaccinationwas associated with the expansion of T cells reactive against tumorneoepitopes (FIG. 11J); however, the neoepitope vaccine is moreefficient at mediating epitope spread than a vaccine targeting thetumor-associated antigen (FIG. 11K).

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their end points, andopen-ended ranges should be interpreted to include commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Furthermore, and unless the context dictatesotherwise, the term “coupled to” is intended to include both directcoupling (in which two elements that are coupled to each other contacteach other) and indirect coupling (in which at least one additionalelement is located between the two elements). Therefore, the terms“coupled to” and “coupled with” are used synonymously.

As used herein, the term “treat” , “treating” or “treatment” of anydisease or disorder refers, in one embodiment, to the administration ofone or more compounds or compositions for the purpose of amelioratingthe disease or disorder (e.g., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof) . In another embodiment “treat”, “treating”, or “treatment”refers to the administration of one or more compounds or compositionsfor the purpose of alleviating or ameliorating at least one physicalparameter including those which may not be discernible by the patient.In yet another embodiment, “treat”, “treating”, or “treatment” refers tothe administration of one or more compounds or compositions for thepurpose of modulating the disease or disorder, either symptomatically,(e.g., stabilization of a discernible symptom), physiologically, (e.g.,breaking the escape phase of cancer immunoediting, induction of anelimination phase of cancer immunoediting, reinstatement of equilibriumphase of cancer immunoediting), or both. In yet another embodiment,“treat”, “treating”, or “treatment” refers to the administration of oneor more compounds or compositions for the purpose of preventing ordelaying the onset or development or progression of the disease ordisorder. The terms “treat”, “treating”, and “treatment” may result, forexample in the case of cancer in the stabilization of the disease,partial, or complete response. However, and especially where the canceris treatment resistant, the terms “treat”, “treating”, and “treatment”do not imply a cure or even partial cure. As also used herein, the term“patient” refers to a human (including adults and children) or othermammal that is diagnosed or suspected to have a disease, and especiallycancer.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C .... andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A composition for use in stimulating an immuneresponse in an individual in need thereof, the composition comprising:a. a first dose of a vaccine composition comprising at least one tumorneoepitope peptide or a nucleic acid encoding at least one tumorneoepitope peptide as a prime vaccination; wherein the at least onetumor neoepitope peptide is determined by 1) whole genome sequencing ofthe individual’s tumor and normal DNA, wherein 9-mer neoepitope peptidesare identified as being encoded by the tumor DNA, 2) selecting from theneoepitope peptides those encoded by RNA with the greatest expressionlevels, and 3) selecting 9-mer neoepitope peptides for predicted bindingaffinity of <500 nM for the HLA-type of the individual; b. IL-15 or aderivative thereof, for use as an immune stimulant to be administeredafter the prime vaccination; c. a checkpoint inhibitor antibody, for useas an immune stimulant to be administered after or concurrently with theIL-15 or derivative thereof; d. a second dose of the vaccinecomposition, for use as an immune stimulant to be administered as aboost vaccination; and e. IL-12, for use as an immune stimulant to beadministered after the boost vaccination.
 2. The composition of claim 1,wherein the prime vaccination and the boost vaccination are between 1and 4 weeks apart.
 3. The composition of claim 1, further comprising aboost vaccination to be administered at least one month after the firstdose of the vaccine composition.
 4. The composition of claim 1, furthercomprising a boost vaccination to be administered at least one yearafter the first dose of the vaccine composition.
 5. The composition ofclaim 1, wherein the administrations of IL-15 or derivative thereof andIL-12 are between 3-5 days apart.
 6. The vaccine composition of claim 1,wherein the nucleic acid encoding the tumor neoepitope peptides is in anexpression vector selected from the group consisting of a bacterialexpression vector, a yeast expression vector, and a viral expressionvector.
 7. The vaccine composition of claim 1, wherein the nucleic acidcomprises an adenoviral vector.
 8. The composition of claim 7, whereinthe adenoviral vector encodes at least four neoepitope peptides.
 9. Thecomposition of claim 1, wherein the checkpoint inhibitor antibodycomprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA-4 inhibitor.10. The composition of claim 9, wherein the checkpoint inhibitorantibody comprises a PD-L1 antibody.
 11. The composition of claim 10,wherein the PD-L1 antibody is selected from the group consisting of10F.9G2, atezolizumab, durvalumab, and avelumab.
 12. The composition ofclaim 1, wherein the IL-15 or derivative thereof comprises an IL-15superagonist.
 13. The composition of claim 12, wherein the IL-15superagonist comprises Alt-803 (N-803).